CN105078966A - Application of cephaloziellin A in preparing medicines in preparing medicine for treating gastric cancer - Google Patents
Application of cephaloziellin A in preparing medicines in preparing medicine for treating gastric cancer Download PDFInfo
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- CN105078966A CN105078966A CN201510540972.4A CN201510540972A CN105078966A CN 105078966 A CN105078966 A CN 105078966A CN 201510540972 A CN201510540972 A CN 201510540972A CN 105078966 A CN105078966 A CN 105078966A
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- cephaloziellin
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Abstract
The invention discloses application of cephaloziellin A in preparing medicines for treating gastric cancer, belonging to the field of medicines. In vitro tests prove that when PDTC and cephaloziellin A separately act on the cancer cells, proliferation of cancer cells can be inhabited, and the cell apoptosis can be promoted; when the two medicines are combined to use, the effect of promoting the cell apoptosis is more obvious, and the growth inhibition rate and the apoptotic index of cells are higher than those of the cells of single medication group. The study finds that by combining the two medicines of the PDTC and the cephaloziellin A to use, the effect of inhibiting the cancer cells is more obvious, and the two medicines are both of non-traditional chemotherapeutic medicines, and are less damage on an organism, and the medicine resistance of tumor cells on traditional chemotherapeutic medicines can be avoided. The cephaloziellin A can be further studied and prepared into the medicines for treating the gastric cancer.
Description
Technical field
The present invention relates to the novelty teabag of Compound C ephaloziellinA, be specifically related to the application of CephaloziellinA in preparation treatment gastric cancer medicament.
Background technology
The people such as Rui-JuanLi separation and purification first goes out Compound C ephaloziellinA, and achievement is published in (SecondaryMetabolitesfromtheChineseLiverwortCephaloziella kiaeri on famous natural product magazine JournalofNaturalProduct, J.Nat.Prod., 2013,76,1700-1708).
Not yet there is the active reporter of this compound at present.
Summary of the invention
The object of the present invention is to provide the medical usage of a kind of CephaloziellinA.
Above-mentioned purpose is achieved by the following technical solution:
The application of CephaloziellinA in preparation treatment gastric cancer medicament, described CephaloziellinA chemical structural formula is as follows,
Further, described gastric cancer is SGC-7901 gastric cancer.
Detailed description of the invention
Essentiality content of the present invention is further illustrated below in conjunction with embodiment.
The separation preparation of embodiment 1:CephaloziellinA and structural identification
The preparation method of CephaloziellinA is with the preparation method (SecondaryMetabolitesfromtheChineseLiverwortCephaloziella kiaeri, J.Nat.Prod., 2013,76,1700-1708) of bibliographical information.
Structural identification: white amorphous powder, molecular formula is C
20h
24o
6, degree of unsaturation is 9.IR spectroscopic data display compound contains hydroxyl (3444cm
-1) and lactone (1770cm
-1) group.Hydrogen nuclear magnetic resonance modal data δ
h(ppm, DMSO-d
6, 600MHz): H-1 (1.93, m), H-1 (2.05, m), H-2 (1.67, m), H-2 (2.89, m), H-3 (4.60, m), H-4 (2.00, br, s), H-6 (4.58, t, J=8.90), H-7 (2.24, dd, J=14.4, 8.4), H-7 (1.77, dd, J=14.4, 9.6), H-10 (2.21, m), H-11 (2.24, dd, J=14.4, 8.4), H-11 (2.30, dd, J=14.4, 6.0), H-12 (5.31, dd, J=8.4, 6.0), H-14 (6.25, br, s), H-15 (7.40, br, s), H-16 (7.38, br, s), H-17 (1.19, s), H-19 (1.39, s), H-20 (5.23, s), carbon-13 nmr spectra data δ
c(ppm, DMSO-d
6, 600Hz): 18.5 (CH
2, 1-C), 25.2 (CH
2, 2-C), 71.9 (CH, 3-C), 55.1 (CH, 4-C), 39.4 (C, 5-C), 84.3 (CH, 6-C), 41.6 (CH
2, 7-C), 73.5 (C, 8-C), 55.8 (C, 9-C), 42.6 (CH, 10-C), 40.3 (CH
2, 11-C), 70.4 (CH, 12-C), 129.1 (C, 13-C), 108.0 (CH, 14-C), 143.8 (CH, 15-C), 138.7 (CH, 16-C), 27.1 (CH
3, 17-C), 178.0 (C, 18-C), 29.9 (CH
3, 19-C), 105.5 (CH, 20-C).
Structural identification data are consistent with bibliographical information, therefore can determine that compound prepared by the present invention is the CephaloziellinA of bibliographical information.
The pharmacological action test of embodiment 2:CephaloziellinA
One, material and instrument
Human stomach cancer cell line SGC-7901 is purchased from Sai Er Reagent Company.CephaloziellinA makes by oneself, and HPLC normalization purity is greater than 98%.PDTC, trypsin, MTT, dimethyl sulfoxide (DMSO), agarose, glacial acetic acid are purchased from Sigma Co., USA.RPMI-1640 culture medium, PBS phosphate buffered solution are purchased from GIBCO company of the U.S..Hyclone is purchased from Shandong Yin Xiang great achievement company.Trypan blue (Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge), TUNEL test kit (Kai Ji biotechnology Development Co., Ltd), DAB colour reagent box (Beijing biotech firm of Zhong Shan Golden Bridge), formaldehyde (Fisher company of the U.S.), catalase (new fine chemistry industry development centre, sky, Tianjin).
WD-9403C uv analyzer (German Biometra company), grads PCR instrument (German Biometra company), Labworks image acquisition and analysis software (Shanghai Tian Neng company), electrophresis apparatus (Beijing 6 1), electronic balance (Shanghai precision instrumentation company limited), RKI-1002 type CO2 gas incubator (Japanese Ikemoto company), superclean bench (Suzhou purification experimental facilities company limited), supercentrifuge (Beijing medical apparatus and instruments factory), low speed centrifuge (Beijing medical apparatus and instruments factory), WilovertS type inverted microscope (Japanese Olympus company), vertical pressure steam sterilizer (Shanghai Bo Xun Industrial Co., Ltd.), cell counting count board (Shanghai precision instrument company), ultra-pure water demineralizer (Britain PURELABulTRAGENETIC).
Two, test method
1, the cultivation of stomach cancer cell SGC-7901
1.1 cell recovery
(1) from liquid nitrogen container, take out cell cryopreservation tube, be placed in rapidly 37 DEG C-42 DEG C, in 75% ethanol, then move in equality of temperature water-bath;
(2) rock l-3min cryopreservation tube gently, cryopreserving liquid is melted, move to rapidly in super-clean bench;
(3) cryopreserving liquid containing cell is sucked sterile centrifugation tube, add appropriate RPIM-1640 culture medium;
(4) put into low speed centrifuge, centrifugal 3 minutes of 800rpm/min after piping and druming mixing, remove supernatant;
(5) adding appropriate culture medium blows even, is inoculated in 10mL culture dish by cell suspension according to concentration;
(6) incubator be placed in containing 5% carbon dioxide, 37 DEG C of saturated humidities is cultivated.
1.2 passage
(1), when the cell attachment in culture dish about 80%, in super-clean bench, cell in several culture dish is blown and beaten, to blow dead cell off by the culture medium that pipette, extract is old;
(2) suck old culture medium with pipet, add appropriate 0.25% trypsin digestion cell, be placed in 30 DEG C of incubators and digest;
(3) observe under culture dish being placed in after about 3min inverted microscope, retraction shinny until cell periphery then illustrates that cell departs from from wall after becoming circle;
(4) in super-clean bench, pancreatin is sucked rapidly.Add appropriate culture medium and repeatedly blow and beat cell, make cell depart from culture dish completely;
(5) cell suspension is seeded in different culture dishs respectively by concentration, supplies culture medium;
(6) be again placed in containing 5%CO
2, 37 DEG C of saturated humidities incubator in cultivate.
2, cell counting
(1) get out cell counting count board and coverslip, the two is all clean by alcohol wipe, is covered by coverslip on counting chamber; (2) after ethanol volatilization, the appropriate cell suspension of sucking-off, drips at coverslip edge, makes suspension be full of between coverslip and counting chamber, notes the glass guide channel not overflowing coverslip and both sides, counting after leaving standstill; (3) find 4 large lattice under the microscope, each large lattice are divided into again 16 little lattice, count the cell number in 4 large lattice respectively, average.On the left of line ball cytometer and top, on the right side of disregarding and below; (4) average cell number × 10000 of cell concentration (individual/mL)=each grid; (5) cell suspension is diluted to experiment desired concn.
3, cell viability measures
(1) SGC-7901 stomach cancer cell suspension 0.9mL to be determined is got; (2) in cell suspension, add trypan blue piping and druming mixing; (3) adopt cell counting count board blind counting at least 200 cells, observe under inverted microscope; (4) Microscopic observation cell dyeing situation, dye light blue person in cell for dead cell, the person of being unstained is living cells, with the vigor (%) of the percentage ratio of total cellular score shared by living cells reflection cell.
4, MTT detects inhibitory rate of cell growth
Experiment grouping: 1. negative control group; 2. CephaloziellinA group 1, CephaloziellinA (50 μm of ol/L); 3. CephaloziellinA2, CephaloziellinA (100 μm of ol/L); 4. PDTC group 1, PDTC (50 μm of ol/L); 5. PDTC group 2, PDTC (100 μm of ol/L); 6. drug combination group 1, CephaloziellinA (25 μm of ol/L)+PDTC (25 μm of ol/L); 7. drug combination group 2, CephaloziellinA (50 μm of ol/L)+PDTC (50 μm of ol/L).
(1) take the logarithm the SGC-7901 cell of trophophase, with cell counting count board counting, adjustment cell concentration is 5 × l0
5/ mL; (2) sample injector is used to be inoculated in 96 orifice plates with every hole 100 μ L.Only be inoculated into 60 middle holes when noting inoculation, periphery 36 hole is filled with PBS, spreads 3 96 orifice plates simultaneously; (3) 96 orifice plates completed are placed in 37 DEG C, 5%CO
2in cell culture incubator, take out after 24h cell attachment; (4) 96 orifice plates are divided into CephaloziellinA group, (0,50,100 μm of ol/L), PDTC group (0,50,100 μm of ol/L), group (0/0,25/25,50/50 μm of ol/L) combined by 2 medicines, sets up not celliferous blank group (only adding culture medium) to give different disposal respectively simultaneously; (5) each concentration of each medicine establishes 5 multiple holes, cultivates 24,48 and 72h respectively; (6) respectively at specific end time point taking-up 96 orifice plate, every hole adds MTT (5g/L) solution 20 μ L, puts back to incubator and continues to cultivate 4h, stop cultivating.(7) carefully suck supernatant in hole, every hole adds 150 μ LDMSO, and plate shaker vibration 10min makes crystallization fully dissolve, and basis of microscopic observation granule disappears.(8) measure optical density (OD) value in each hole in microplate reader 490nm wavelength place, calculate the inhibitory rate of cell growth of each time point.Suppression ratio=[1-(dosing group OD value-blank group OD value)/(negative control group OD value-blank group OD value)] × 100%.
5, TUNEL method detects natural death of cerebral cells index
The preparation of 5.1 cell climbing sheets
(1) process of coverslip: coverslip is placed in concentrated sulphuric acid soaked overnight, next day first with tap water for several times, then be placed in dehydrated alcohol and soak 4h, then clean with deionized water rinsing, be placed on for drying the sterilization of laggard horizontal high voltage in dry case, it is for subsequent use that super-clean bench is directly put in taking-up.6 orifice plates are placed in ultraviolet radiation 30min in super-clean bench;
(2) coverslip is placed: after the position preparing to put coverslip in every hole of six orifice plates instills a small amount of culture medium, place coverslip again, the tension force making coverslip and orifice plate examine culture medium is bonded together, when preventing from adding cell suspension, coverslip hikes up, and causes double-layer cell adherent;
(3) take the logarithm the SGC-7901 cell of trophophase, cell suspension is blown and beaten in digestion, is l × 10 with cell counting count board adjustment cell concentration
6/ mL.
(4) add 1mL cell suspension respectively with every hole that sample injector is being placed with coverslip, spread 3 plates simultaneously, be placed in 37 DEG C, 5%CO
2cultivate 24h in incubator and treat cell attachment;
In super-clean bench, negative control group and experimental group is divided into give different disposal respectively 6 orifice plates after (5) 24 hour cells are adherent, negative control group adds 1mL culture medium, experimental group is further divided into CephaloziellinA group, PDTC group and drug combination group 3 subgroups, and every hole adds 1mL medicine.CephaloziellinA group final concentration is 100 μm of ol/L, PDTC group final concentrations is 100 μm of ol/L, 2 medicines combine group final concentration to be 2 kinds of medicines be respectively 50 μm of ol/L often group establish 2 multiple holes.
(6) 37 DEG C are placed in, 5%CO
2cell culture incubator in continue cultivate.
5.2TUNEL method operating procedure
(1) take out 6 orifice plates when 24h is cultivated in cell climbing sheet dosing, carry out following steps successively according to TUNEL description; (2) supernatant abandoned in every hole is carefully inhaled; (3) each hole adds PBS (4 DEG C) 2mL, and plate shaker rinses 5min × 3 time; (4) every hole adds 1mL pre-cooling cell fixative, and 30min fixed by 4 DEG C of refrigerators.(5) fixative is abandoned in suction, and each hole adds PBS (4 DEG C) 2mL, and plate shaker rinses 5min × 3 time.(6) immerse in confining liquid, room temperature (15 DEG C-25 DEG C) closes 10min.(7) each hole adds PBS (4 DEG C) 2mL, and plate shaker rinses 5min × 3 time.Blot with absorbent paper around sample.(8) each sample drips 50 μ LUTdT enzyme reaction solutions, 37 DEG C, lucifuge moistening reaction 60min.(9) each hole adds PBS (4 DEG C) 2mL, and plate shaker rinses 5min × 3 time.Blot with absorbent paper around sample.(10) 50 μ LStreptavidin-HRP working solutions are dripped, 37 DEG C, lucifuge moistening reaction 30min.(11) each hole adds PBS (4 DEG C) 2mL, and plate shaker rinses 5min × 3 time.(12) 100 μ LDAB working solutions are dripped, color development at room temperature reaction 10min.(13) each hole adds PBS (4 DEG C) 2mL, and plate shaker rinses 5min × 3 time.(14) optical microphotograph Microscopic observation is taken pictures: random selecting 10 high power (× 100) visuals field, and each visual field counts 100 cells, calculates the meansigma methods adjusting index of dying.Apoptotic index (AI)=(positive cell number/total cell number × 100%).The nucleus of positive cell is sepia.
6, statistical analysis
Adopt SPSS11.5 to analyze, the measurement data meeting the distribution of JH state, to represent, compares between group and analyzes by Oneway-ANOVA method, and compare between two and adopt LSD-t method, P<0.05 is that difference has statistical significance.
Three, result and conclusion
1, MTT detection of drugs is to the growth inhibition ratio of SGC-7901 stomach cancer cell
MTT is repeated through 3 times, testing result shows, single drug effect is after stomach cancer cell 72h, and the medicine group of 100 μm of ol/L is all higher to the growth inhibition ratio of stomach cancer cell compared with the medicine group of 50 μm of ol/L, and difference has statistical significance (P<0.05).The CephaloziellinA of 50 μm of ol/L is to the growth inhibited effect of stomach cancer cell and drug combination 25/25 μm of ol/L group no significant difference (P>0.05).The PDTC of 50 μm of ol/L is to compared with the growth inhibited effect of stomach cancer cell and drug combination 25/25 μm of ol/L group, and difference has statistical significance (P<0.05).Drug combination 50/50 μm of ol/L group to the growth inhibition ratio of stomach cancer cell higher than each high concentration list medicine group (100 μm of ol/L) growth inhibition ratio to stomach cancer cell, difference has statistical significance (P<0.001), in table 1 (* P<0.05, * * P<0.01).
2, TUNEL method detects each group of apoptotic index
CephaloziellinA, PDTC and drug combination group all have the effect of Developing restraint to stomach cancer cell SGC-7901, each group all has statistical significance (P<0.001) with negative control group comparing difference, negative control group has the karyon of a small amount of cell to be dyed to sepia, each single medicine group and drug combination group apoptotic cell comparatively all have increase compared with negative control group, the apoptotic index of drug combination group cell is the highest, more remarkable to the inhibition of stomach cancer cell.In table 2 (* * P<0.01).
Conclusion, PDTC and CephaloziellinA acts solely on stomach cancer cell all can the propagation of anticancer, promote the apoptosis of cell, along with the increase of concentration, be enhancing trend to the Developing restraint effect of stomach cancer cell, promote the apoptosis more remarkable effect of stomach cancer cell after 2 kinds of Drug combinations, all more independent medication group of the growth inhibition ratio of cell and apoptotic index raises.Research finds that the inhibitory action of PDTC and CephaloziellinA2 kind Drug combination to stomach cancer cell is more remarkable, and 2 kinds of medicines are the chemotherapeutics of non-traditional meaning, little to the infringement of body, and avoid the drug resistance of tumor cell to classic chemotherapy medicine, reference can be provided for the clinical treatment of gastric cancer from now on.
Table 1 each medicine group different time points to the growth inhibition ratio of stomach cancer cell (n=5,
)
| Group | n | 24h | 48h | 72h |
| Cephaloziellin A 50μmol/L(1) | 5 | 14.87±5.19 | 16.29±4.53 | 55.70±6.67 |
| Cephaloziellin A 50μmol/L(2) | 5 | 31.00±6.36 | 59.42±7.74 | 74.32±5.84 |
| PDTC 50μmol/L(3) | 5 | 24.87±9.04 | 36.83±5.21 | 40.58±7.15 |
| PDTC 100μmol/L(4) | 5 | 42.86±6.28 | 44.21±8.44 | 50.31±4.63 |
| Drug combination 25/25 μm of ol/L (5) | 5 | 36.43±8.13 | 38.76±10.87 | 58.55±9.53 |
| Drug combination 50/50 μm of ol/L (6) | 5 | 49.38±2.24 | 74.56±10.59 | 96.12±2.25 |
| F | 17.918 ** | 29.646 ** | 47.625 ** | |
| P(1):(2) | 0.001 | <0.001 | <0.001 | |
| (3):(4) | <0.001 | 0.169 | 0.025 | |
| (5):(6) | 0.005 | <0.001 | <0.001 | |
| (1):(5) | <0.001 | <0.001 | 0.49 | |
| (3):(5) | 0.01 | 0.713 | <0.001 | |
| (2):(6) | <0.001 | 0.008 | <0.001 | |
| (4):(6) | 0.13 | <0.001 | <0.001 |
After table 2 drug effect 24h each group cell apoptotic index (n=3,
)
Claims (2)
- The application of 1.CephaloziellinA in preparation treatment gastric cancer medicament, described CephaloziellinA chemical structural formula is as follows,
- 2. the application of CephaloziellinA according to claim 1 in preparation treatment gastric cancer medicament, is characterized in that: described gastric cancer is SGC-7901 gastric cancer.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105085452A (en) * | 2015-09-05 | 2015-11-25 | 林天样 | Sesquiterpene naphthoquinone compound as well as preparation method and medical application thereof |
| CN105520928A (en) * | 2015-12-28 | 2016-04-27 | 吴芊葭 | Application of diterpene compound in preparation of breast cancer inhibition drugs |
| CN105585574A (en) * | 2015-12-28 | 2016-05-18 | 吴芊葭 | Diterpene compound and preparation method thereof |
| WO2017071380A1 (en) * | 2015-10-28 | 2017-05-04 | 李淑兰 | Tumor vaccine for use in treating liver cancer and preparation method for vaccine |
-
2015
- 2015-08-28 CN CN201510540972.4A patent/CN105078966A/en not_active Withdrawn
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105085452A (en) * | 2015-09-05 | 2015-11-25 | 林天样 | Sesquiterpene naphthoquinone compound as well as preparation method and medical application thereof |
| WO2017071380A1 (en) * | 2015-10-28 | 2017-05-04 | 李淑兰 | Tumor vaccine for use in treating liver cancer and preparation method for vaccine |
| CN105520928A (en) * | 2015-12-28 | 2016-04-27 | 吴芊葭 | Application of diterpene compound in preparation of breast cancer inhibition drugs |
| CN105585574A (en) * | 2015-12-28 | 2016-05-18 | 吴芊葭 | Diterpene compound and preparation method thereof |
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Application publication date: 20151125 |